Timing molecular motion and production with a synthetic transcriptional clock

The realization of artificial biochemical reaction networks with unique functionality is one of the main challenges for the development of synthetic biology. Due to the reduced number of components, biochemical circuits constructed in vitro promise to be more amenable to systematic design and quantitative assessment than circuits embedded within living organisms. To make good on that promise, effective methods for composing subsystems into larger systems are needed. Here we used an artificial biochemical oscillator based on in vitro transcription and RNA degradation reactions to drive a variety of “load” processes such as the operation of a DNA-based nanomechanical device (“DNA tweezers”) or the production of a functional RNA molecule (an aptamer for malachite green). We implemented several mechanisms for coupling the load processes to the oscillator circuit and compared them based on how much the load affected the frequency and amplitude of the core oscillator, and how much of the load was effectively driven. Based on heuristic insights and computational modeling, an “insulator circuit” was developed, which strongly reduced the detrimental influence of the load on the oscillator circuit. Understanding how to design effective insulation between biochemical subsystems will be critical for the synthesis of larger and more complex systems

Modular chemical mechanism predicts spatiotemporal dynamics of initiation in the complex network of hemostasis

This article demonstrates that a simple chemical model system, built by using a modular approach, may be used to predict the spatiotemporal dynamics of initiation of blood clotting in the complex network of hemostasis. Microfluidics was used to create in vitro environments that expose both the complex network and the model system to surfaces patterned with patches presenting clotting stimuli. Both systems displayed a threshold response, with clotting initiating only on isolated patches larger than a threshold size. The magnitude of the threshold patch size for both systems was described by the Damköhler number, measuring competition of reaction and diffusion. Reaction produces activators at the patch, and diffusion removes activators from the patch. The chemical model made additional predictions that were validated experimentally with human blood plasma. These experiments show that blood can be exposed to significant amounts of clot-inducing stimuli, such as tissue factor, without initiating clotting. Overall, these results demonstrate that such chemical model systems, implemented with microfluidics, may be used to predict spatiotemporal dynamics of complex biochemical networks